Connective tissue growth factor (CTGF) is a growth factor expressed by endothelial and fibroblastic cells. Two members of the CTGF family are known, CTGF (U.S. Pat. Nos. 5,585,270 and 5,408,040) and CTFG-2 (WO 96/01896), incorporated herein by reference. CTGF belongs to a family of growth factors that includes CTGF, CTGF-2, insulin binding proteins (IBP) 1 and 2. These growth factors have a cysteine-rich motif and several other structural protein domains in common, and have been demonstrated to have a role in cell proliferation, differentiation, and chemotaxis.
Studies have suggested that CTGF is immunologically similar to PDGF, with PDGF antibodies from both A and B chains binding to CTGF (Bradham et al., J. of Cell Biol. 114 (6):1285-1294, 1991), and it has been reported that biological activity of CTGF can be blocked using these antibodies.
It has recently been demonstrated that porcine CTGF isolated from uterus is mitogenic for fibroblasts and smooth muscle cells but not endothelial cells. In addition, a N-terminally truncated form of the protein was active as well (Brigstock et al., J. Biol. Chem. 272:20275-20282, 1997). It has been suggested that this protein could play a role in the growth and remodeling of the endometrium and placenta.
The CTGF family is believed to play a role production of the extracellular matrix components, such as collagen and fibronectin. Collagen and fibronectin are components of many connective tissues, e.g., ligaments, cartilage, tendons and vessel walls. Current therapy for ligament repair is limited to immobilising the damaged tissue, i.e., stapling, or replacing the damaged tissue with synthetic or natural grafts. These tissues are notoriously difficult to heal, even in healthy individuals, and compositions that would improve recovery time would be very valuable.
The present invention is directed to novel polypeptides and polynucleotides encoding the polypeptide that show predominantly high expression in testis, trachea and bone marrow, thus providing a new molecule for regulating growth, differentiation, chemotaxis and induction of specialized cell functions in these tissues.
It is an object of the present invention to provide an isolated polynucleotide molecule comprising a polynucleotide sequence that encodes a connective tissue growth factor homolog polypeptide that is at least 70% identical to the amino acid sequence as shown in SEQ ID NO: 2 from residue 24 to residue 354.
In another embodiment, the present invention provides for a polynucleotide molecule wherein the polynucleotide molecule comprises a region having the following motif as shown in SEQ ID NO: 23:
Cx{8,10}CxCCxxCx{7}Cx{5,6}Cx{5,7}Cx{12,13}Cx{7,8}Cx{20}CxCx{6}Cx{12,14}Cx{13,17}C
wherein x{ } is the number of amino acid residues between cysteines (C).
In another embodiment, the polynucleotide is 80% or 90% identical to the amino acid sequence as shown in SEQ ID NO: 2 from residue 24 to residue 354.
It is also the object of the present invention to provide an isolated polynucleotide acid molecule that encodes a connective tissue growth factor homolog polypeptide, wherein the polynucleotide molecule is selected from the group consisting of (a) a molecules having the nucleotide sequence of SEQ ID NO:1 from nucleotide 17 or 86 to nucleotide 1078, (b) a molecule encoding the amino acid sequence of SEQ ID NO:3 from nucleotide 1 or 70 to nucleotide 1062, and (c) a molecule that hybridizes under stringent wash conditions to a polynucleotide molecule having the nucleotide sequence of nucleotides 86 to 1078 of SEQ ID NO:1, or the complement of nucleotides 86 to 1078 of SEQ ID NO:1.
In another embodiment, the differences in the amino acid sequence encoded by the polynucleotide and SEQ ID NO: 2 are conservative amino acid changes.
In other aspects, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment comprising the isolated polynucleotide sequence that encodes a connective tissue growth factor homolog polypeptide that is at least 70% identical to the amino acid sequence as shown in SEQ ID NO: 2 from residue 24 to residue 354; and a transcription terminator and a cultured host cell into which has been introduced the expression vector.
In another embodiment, the present invention provides a method of producing a connective tissue growth factor homolog polypeptide comprising: (a) culturing the host cells of expressing the CTGF homolog polypeptide; and (b) isolating the connective tissue growth factor homolog polypeptide from the cultured host cells.
In another aspect, the present invention provides an isolated connective tissue growth factor polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence as shown in SEQ ID NO: 2 from residue 24 to residue 354.
In other embodiments, the present invention provides CTGF homolog polypeptides wherein the amino acid sequence is at least 80% or 90% identical.
In another embodiment, the CTGF homolog polypeptide molecule comprises a region having the following motif as shown in SEQ ID NO: 23:
Cx{8,10}CxCCxxCx{7}Cx{5,6}Cx{5,7}Cx{12,13}Cx{7,8}Cx{20}CxCx{6}Cx{12,14}Cx{13,17}C
wherein x{ } is the number of amino acid residues between cysteines (C).
In another aspect, the present invention provides an antibody or antibody fragment that specifically binds with the CTGF homolog polypeptide.
In another aspect, the present invention provides a method of detecting the presence of connective tissue growth factor homolog polypeptide in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody, or an antibody fragment of claim 14, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.
In another aspect, the present invention provides an anti-idiotype antibody, or anti-idiotype antibody fragment, that specifically binds with the antibody or antibody fragment.
In another aspect, the present invention provides a method of detecting a chromosome 6q abnormality in sample from an individual comprising: (a) obtaining zCTGF4 RNA from the sample; (b) generating zCTGF4 cDNA by polymerase chain reaction; and (c) comparing the nucleic acid sequence of the zCTGF4 cDNA to the nucleic acid sequence as shown in SEQ ID NO: 1.
In another embodiment, the present invention provides that the difference between the sequence of the zCTGF4 cDNA or zCTGF4 gene in the sample and the zCTGF4 sequence as shown in SEQ ID NO: 1 is indicative of chromosome 6q abnormality.